Abstract Though immunosuppressive agents (ISA) used to prevent rejection have improved over time, they are still unable to consistently eliminate acute and chronic rejection and they have been implicated in the pathogenesis of organ failure (i.e., calcineurin inhibitor toxicity). One of the main issues with traditional ISA is that they inhibit the regulatory pathways in transplantation as well. Therefore, one of the highest unmet needs in transplantation is to identify novel immunomodulatory strategies which selectively inhibit the pathogenic alloreactive immune cells but spare regulatory humoral and cellular pathways such as T regulatory cells (Tregs). Phosphoinositide-3 kinases (PI3K) are lipid kinases that generate critically important messengers for immune cell function. Among the various subclasses of PI3K, PI3K? has received much attention for its restricted expression in leukocytes and regulatory role in immune cells and inflammation. Nevertheless, the importance of targeting PI3K? signaling pathways in transplantation remains unexplored. We have generated a substantial amount of data indicating that PI3K? inhibition markedly reduces acute heart transplant rejection. Notably, there was marked synergism between PI3K? inhibition and low dose CTLA4-Ig which resulted in indefinite prolongation of heart allograft survival. Mechanistically, PI3K? inhibition resulted in selective suppression of T effector cells (Teff) while augmenting Tregs. Our mechanistic data also indicate that PI3K? inhibition activates genes known to promote FOXP3 promoter. PI3K? inhibition markedly reduced the production of inflammatory cytokines which induce alloimmunity. Our overall objective is to decipher the relative contribution of the PI3K? subclass in T cell-dependent alloimmunity and use the new information to support the discovery of therapeutics that have high potential to enhance immunoregulation in alloimmunity. We hypothesize that by tipping the balance of Teff/Tregs toward Tregs, PI3K? inhibition effectively suppresses transplant rejection. In Aim 1, we will examine the role of pharmacological inhibition of PI3K? in suppressing both acute and chronic rejection. In Aim 2, we will examine the mechanisms by which PI3K? inhibition suppresses alloimmune responses. We will also study the impact of PI3K? inhibition on alloantigen specific Tregs. In Aim 3, we will study the synergism of inhibiting both the PI3K? and B7-CD28 pathways in tolerance induction. Alloimmune responses are critically determined by the balance of Teff and Tregs. Identifying novel regulatory pathways which control this balance has a significant impact on the design of future immunomodulatory therapies in transplantation. These studies will provide critical information on the role of PI3K? in controlling the balance of Teff/Tregs. These data can serve as a basis for future PI3K?-based therapies for transplantation.